Technology (BMTi)
Committee members
Prof. dr.‐ing. M. Wessling promotor University of Twente
Dr. D. Stamatialis assistant promotor University of Twente
Prof. dr. K. Boller chairman University of Twente
Prof. dr. C.A. van Blitterswijk University of Twente Prof. dr. D.W. Grijpma University of Twente, University of Groningen Prof. D. Kaplan Tufts University, USA Prof. A. Boccaccini Imperial College London, UK Prof. dr. R.A Bank University Medical Center Groningen © 2009 Bernke Papenburg, Enschede, The Netherlands All rights reserved Design Strategies for Tissue Engineering Scaffolds Bernke Papenburg PhD thesis, University of Twente, The Netherlands ISBN: 978‐94‐9012‐239‐3 Printed by: Gildeprint Drukkerijen, Enschede, The Netherlands Cover design by Bernke Papenburg The cover shows a fluorescence microscopy image of pre‐myoblast cells organized on a poly(L‐lactic acid) scaffold featuring pillars; green indicates the cytoskeleton of the cells whereas the nuclei are labeled blue
DESIGN STRATEGIES FOR TISSUE ENGINEERING SCAFFOLDS
PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit Twente, op gezag van de rector magnificus, prof. dr. H. Brinksma volgens besluit van het College voor Promoties in het openbaar te verdedigen op vrijdag 19 juni 2009 om 13.15 uur door Berendien Jacoba Papenburg Geboren op 22 juli 1981 te Dedemsvaart
Promotor: Prof. Dr.‐Ing. M. Wessling en Assistent promotor: Dr. D. Stamatialis
Voor mijn oma, Berendina Hartkamp, die dit proefschrift nog zo graag zelf had ingezien “Dancing in all its forms cannot be excluded from the curriculum of all noble education; dancing with the feet, with ideas, with words, and, need I add that one must also be able to dance with the pen?” Friedrich Nietzsche
Chapter 1 General Introduction
1
1.1 Tissue engineering 1 1.2 Tissue engineering in the clinic 3 1.3 Scope and outline of the thesis 4 1.3.1 Scope of the project at large 4 1.3.2 Scope thesis 4 1.4 Outline thesis 8 References 10Chapter 2 Scaffolds for Tissue Engineering
11
2.1 Requirements scaffold 11 2.2 Materials 12 2.2.1 Common biomaterials 12 2.2.2 Poly(lactic acid) 13 2.2.3 Poly(ε‐caprolactone) 14 2.2.4 Poly(tri‐methylene carbonate) 14 2.2.5 Poly(ethylene oxide)/poly(butylene terephtalate) 15 2.2.6 Poly(dimethyl siloxane) 15 2.3 Fabrication methods 16 2.3.1 Emulsion freeze‐drying 16 2.3.2 Foaming 16 2.3.3 Particle leaching 17 2.3.4 Electrospinning 17 2.3.5 Sintering 18 2.3.6 Polymer casting and phase separation 18 2.4 Scaffold design 20 References 21to Control Cell Behavior
27
3.1 Introduction 28 3.2 Background phase separation micromolding (PSµM) 30 3.3 Materials and Methods 31 3.3.1 Micropatterned porous scaffold preparation 31 3.3.2 Porosity and pore morphology determination 32 3.3.3 Nutrient transport 33 3.3.4 Cell culturing 34 3.3.5 3‐D scaffolds proof‐of‐principle 36 3.4 Results and Discussion 37 3.4.1 Micropatterned sheet preparation 37 3.4.2 Porosity and pore morphology determination 37 3.4.3 Nutrient transport 40 3.4.4 Cell culturing 42 3.4.5 3‐D scaffolds proof‐of‐principle 47 3.5 Conclusions 48 References 49Chapter 4 A Facile Method to Fabricate Poly(L‐lactide) Nano‐fibrous
Morphologies by Phase Inversion
51
4.1 Introduction 52 4.2 Materials and Methods 53 4.2.1 PLLA synthesis and characterization 53 4.2.2 Scaffold preparation and characterization 53 4.2.3 Cell culturing and analysis 54 4.3 Results and Discussion 56 4.3.1 PLLA synthesis and characterization 56 4.3.2 Scaffold preparation 56 4.3.3 Scaffold characterization 57 4.3.4 Cell culture and analysis 62 4.4 Conclusions 65 References 66Poly(1,3‐trimethylene carbonate) Scaffolds
69
5.1 Introduction 70 5.2 Materials and Methods 71 5.2.1 PTMC synthesis and characterization 71 5.2.2 Scaffold preparation 72 5.2.3 PEO content 74 5.2.4 Porosity and pore morphology 74 5.2.5 Sheet morphology under physiological conditions 75 5.2.6 Cell culturing 75 5.2.7 Cell culture analysis 75 5.3 Results and Discussion 77 5.3.1 PTMC synthesis and characterization 77 5.3.2 PTMC scaffold preparation 77 5.3.3 Porosity and pore morphology 79 5.3.4 Sheet morphology under physiological conditions 82 5.3.5 Cell morphology 84 5.3.6 Cell proliferation 85 5.4 Conclusions 92 References 93Chapter 6 Cell Behavior as Function of Contact Angle and Surface
Topography
95
6.1 Introduction 96 6.2 Materials and Methods 98 6.2.1 Topography design 98 6.2.2 Micropatterned scaffold sheet preparation and characterization 98 6.2.3 Cell culturing and analysis 100 6.3 Results and Discussion 101 6.3.1 Micropatterned scaffold sheet preparation 101 6.3.2 Contact angle ‐ wettability 103 6.3.3 Protein adsorption 106 6.3.4 Cell attachment and proliferation 108 6.3.5 Cell morphology 111 6.4 Conclusions 117 References 118Interactions
123
7.1 Introduction 124 7.2 Concept 125 7.2.1 Paradigm 125 7.2.2 Design 127 7.3 Materials and Methods 128 7.3.1 Mask design and mold fabrication 128 7.3.2 TopoChip fabrication 128 7.3.3 TopoChip seeding and culturing 129 7.4 Results and Discussion 130 7.4.1 TopoChip fabrication 130 7.4.2 Uniform seeding 132 7.4.3 Cell‐material interactions 134 7.5 Conclusions 136 References 137Chapter 8 Understanding Nutrient Supply through Multi‐Layer
Scaffolds
139
8.1 Introduction 140 8.2 Model 143 8.2.1 Concept 143 8.3 Materials and Methods 144 8.3.1 Scaffold sheets preparation 144 8.3.2 Porosity and pore morphology determination 144 8.3.3 Cell culturing 145 8.4 Results and Discussion 148 8.4.1 Scaffold sheets characterization 148 8.4.2 Nutrient transport limitations 149 8.4.3 Static culture conditions: multi‐layer WS scaffolds 150 8.4.4 Static culture conditions: multi‐layer TS scaffolds 154 8.4.5 Model results and validation 157 8.4.6 Dynamic culture conditions 160 8.5 Conclusions 165 References 166Appendix to Chapter 8 Model description
169
9.1 Scaffold design at large 177 9.2 Scaffold design in this thesis 178 9.2.1 General discussion 178 9.2.2 General conclusions 179 9.3 Future directions 180 9.3.1 Materials properties: reinforcements 180 9.3.2 Surface characteristics: layer‐by‐layer architecture 181 9.3.3 3D‐architecture: nutrient supply in‐vivo style 181 References 184
Summary
185
Nederlandse Samenvatting
188
Acknowledgments ‐ Dankwoord
192
Cirruculum Vitae ‐ List of Publications
195
1.1 Tissue engineering
The replacement of organs since long has been the subject of debate, however, the field of engineering tissue in vitro to repair damaged tissue in vivo originated only about two decades ago [1, 2]. In fact, tissue engineering (TE) originates from reconstructive surgery where direct transplantation of (allogenic) donor tissue is practiced to repair the function of damaged tissue. Many difficulties arise with direct transplantation due to insufficient donor organs, pathogen transmission and rejection of the donor organ [3‐5]. As a result, patients can be waiting for a donor organ for years, and when they receive one in time, they need to take immunosuppressive medication for the rest of their lives and risk the need of a replacement organ within days to years after the surgery. An autogenic tissue engineering transplant (using patient’s own cells) would address most limitations of direct transplantation and avoid difficulties concerning rejection and pathogen transmission. Additionally, there would be no dependency on donors. Therefore, constructing a tissue engineered replacement in vitro is considered an excellent alternative to direct transplantation of donor organs [1, 3‐5].
TE is defined as the interdisciplinary field applying the principles and methods of engineering and life sciences to fundamentally understand and develop biological substitutes to restore, maintain or improve tissue functions [1]. In basis, TE attempts to mimic the function of natural tissue. Therefore, to optimize the development of functional biological substitutes, the natural circumstances of the specific tissue have to be fundamentally understood. Biological tissues basically consist of cells, signaling systems and extracellular matrix (ECM) [4]. The cells are the core of the tissue, however, can not function in the absence of signaling systems and/or of the ECM. The signaling system consists of genes that secrete transcriptional products when differentially activated, and urges cues for tissue formation and differentiation [4]. The ECM is a meshwork‐like substance within the extracellular space and supports cell attachment and promotes cell proliferation [6, 7].
TE approaches can generally be sub‐divided based on these 3 phenomena, either studied single or combined [1, 3]: - cell‐based therapies - induction of tissue‐formation by soluble signaling factors - and/or biocompatible support by an artificial ECM (scaffold) Figure 1 Schematic illustration of the tissue engineering principle.
Figure 1 illustrates the basic principle of the general TE approach based on the introduction of a scaffold. Cells are isolated from either the patient (autogenic) or from a donor (allogenic). After isolation, the cells are cultured in vitro and subsequently introduced to a scaffold. Finally, the cell‐ cultured scaffold is implanted in vivo into the patient. Many aspects in this process add to the final suitability and functionality of the tissue engineered construct. Isolation of the cells and in vitro culturing requires optimal processing and environmental conditions, e.g. pH, temperature, medium composition. Each type of tissue requires distinct conditions and therefore, demands the understanding of the specific natural biological environment in vivo to allow optimization of culturing in vitro. Supplementing tissue‐inducing soluble factors can be combined within the scaffold‐based approach to guide cell behavior by triggering specific reactions through pathway activation [8‐10]. Another approach to guide cell behavior lies within the architectural design of the scaffold. The scaffold should allow tissue formation in 3D by good support of cell attachment, proliferation and organization, as well as enabling sufficient nutrient supply to the cells and waste elimination from the cells [6, 11, 12]. Design of well‐functioning tissue engineered constructs requires optimization of these aspects for the specific application.
Ch ap te r 1
1.2 Tissue engineering in the clinic
A well‐known example of tissue engineering is the ‘mouse with the human ear’ [13]. This study describes seeding of chondrocytes isolated from bovine articular cartilage on a polymer (polyglycolic acid‐polylactic acid) scaffold in the form of a human ear, and subsequent transplantation of the construct subcutaneous in a nude mouse. Even though the scaffold had the form of a human ear, actually no human material was involved; this study attempted to demonstrate growth of new cartilage in a specific form to be used in plastic or reconstructive surgery. And they did, new cartilage formed within 12 weeks post‐implantation.
With respect to human application, successful clinical trials of tissue engineering constructs have been reported in literature. Nowadays, major areas of tissue‐engineered replacements in clinical trials and applications are skin, cardiovascular, bone and cartilage [5, 7, 14‐16]. One example is the work performed by Matsumura et al. [17] who reported application of tissue‐engineered autografts in cardiovascular surgery on children with various complex heart diseases. They applied a tissue engineering technique where patients own (autogenic) cells were isolated, cultured and subsequently seeded on a biodegradable polymer scaffold of poly(glycolic acid) combined with poly(lactic acid‐ε‐caprolactone). The first operation was performed in May 1999, and over 40 patients were treated the following years. Post‐operative analysis revealed no complications related to the tissue engineering autograft.
Another recent example is the design and implantation of a tissue‐engineered airway [18]. In this case, the researchers first removed all donor cell and antigens of an allogenic donor trachea (wind‐ pipe) to prevent an immune reaction of the host towards the donor material. Subsequently, they re‐ cultured the matrix with autogenic cells and transplanted the cell‐seeded scaffold into the patient’s main bronchus. Immediately, the tissue engineered trachea became functional and after 4 months, the scaffold still showed normal appearance and good mechanical properties.
1.3 Scope and outline of the thesis
1.3.1 Scope of the project at large
Within the definition of tissue engineering lays the need of an interdisciplinary approach and therewith, the collaboration of experts of all participating disciplines. To facilitate collaboration within the interdisciplinary fields combined in (bio)medical research at large, the University of Twente initiated the Institute of Biomedical Engineering (BMTi). The work presented in this thesis is performed within the spearhead program of the BMTi ‘Advanced Polymeric Microstructures for Tissue Engineering’. This project aimed at strengthening the collaboration between the departments of Membrane Science and Technology (MST), Polymer Chemistry & Biomaterials (PBM) and Biophysical Engineering (BPE). With this in mind, several collaborations between these departments followed resulting in a number of papers presenting work of the combined disciplines. Additionally, good collaboration was initiated with the another member of BMTi, the department of Tissue Regeneration (TR), and all experiments involving cell culturing described in this thesis were performed in collaboration with TR.
1.3.2 Scope thesis
This thesis focuses on various aspects involved in scaffold design and the interaction of scaffolds with the cells. The ultimate goal is to design a scaffold that supports functional tissue formation, resembling in vivo tissue organization, combined with good nutrient supply to the cells. Our concept to reach this goal is based on 3D multi‐layer scaffolds consisting of porous micropatterned sheets. Figure 2 illustrates this concept, where cells grow within micropatterned channels giving a clear direction to the cells inducing cell organization (Figure 2a). Subsequent stacking of these micropatterned porous sheets (Figure 2b) results in a 3D scaffold where the microchannels also provide space allowing perfusion of nutrients throughout the complete scaffold [19]. Additionally, the porosity of the single layers enables diffusion of nutrients and signalling factors between the layers.
Ch ap te r 1 Figure 2 Schematic illustration of the 3D multi‐layer scaffold concept. To produce functional scaffolds based on this concept, many parameters need to be understood and optimized regarding the role of scaffold design in the cell‐scaffold interaction at various levels. The work described in this thesis aims at acquiring insight into the role of a number of these aspects and how those affect cell‐material interaction. At large, scaffold design aspects can be divided into three main classes: material properties, surface characteristics and 3D architecture. These three main classes are again subdivided into many sub‐categories, as schematically represented in Figure 3.
This thesis aims to fill in which topics make up the sub‐categories within scaffold design and which parameters are involved when addressing these topics.
Figure 3 Data tree representing the main scaffold design classes.
Nonetheless, this thesis cannot address all sub‐categories within the large scaffold design framework, hence; a selection of topics is evaluated regarding the main design concept of a multi‐layer 3D scaffold incorporating porosity as well as topography. Certain topics within this concept at large are studied in more detail, amongst which material processing, scaffold‐cell interaction regarding surface topography and nutrient supply through this multi‐layer scaffold. Figure 4 schematically illustrates these topics studied in more detail and how these topics mutually interact, in order to obtain the final goal. In fact each topic, originating from the either the engineering, life sciences or the interdisciplinary field in between, has to be studied properly to successfully convert a scaffold from material into part of a TE construct.
Figure 4 Representation of various topics regarding scaffold design and their mutual relations, addressed in this thesis.
Ch ap te r 1 Parameters involved within these addressed topics are e.g.: - Material science: material nature and processing - Scaffold architecture: surface topography design, porosity, size
- Scaffold‐cell interaction: cell attachment, cell proliferation, cell morphology regarding distinct surface characteristics
- Up‐scaling: high‐throughput screening of variables, top‐down design - Going 3D: multi‐layer stacking methods, culturing in 3D
- Nutrient supply: mass transport through layers, perfusion of the channels, theoretical modelling
Even though this thesis cannot address each topic involved within the large scaffold design framework, this thesis aims at providing distinct strategies for functional scaffold design as part of the development of tissue engineering constructs.
1.4 Outline thesis
Whereas this first Chapter presents a general overview of the tissue engineering field, Chapter 2 gives a more detailed literature overview of various aspects in scaffold design. Topics discussed are the general requirements involved in scaffold design, well‐known and frequently used biomaterials, commonly applied scaffold fabrication methods and the importance of surface topography. Additionally, these sections highlight the specific biomaterials and fabrication method adopted in the various chapters of this thesis.
The method used to fabricate the micropatterned porous scaffolds, as illustrated in Figure 2, is Phase Separation Micromolding (PSµM). Chapter 3 offers an overall description of our approach and is related to the vast majority of the topics addressed in this thesis, i.e. materials science, scaffold architecture, scaffold‐cell interaction, going 3‐D as well as nutrient supply. This chapter is dedicated to the application of PSµM in scaffold fabrication and how to tune the morphology of the scaffolds by variation in processing parameters. Application of different polymers is evaluated, followed by a more detailed study of the most porous poly(L‐lactic acid) (PLLA) scaffold sheets with respect to nutrient diffusion through the inner‐porosity of the sheets. Additionally, this chapter demonstrates pre‐myoblast cells (C2C12) alignment tuned by distinct micropatterns and finally, a proof‐of‐principle for stacking of the micropatterned sheets into a 3D scaffold.
Chapter 4 and Chapter 5 more specifically relate to materials science and the corresponding scaffold‐ cell interaction. Chapter 4 presents the effect of the molecular weight of PLLA on the porous sheet morphology during phase separation processing. Very high molecular weight PLLA yields a nano‐ fibrous morphology in contrast to a solid‐wall pore morphology generally obtained using lower molecular weight PLLA, described in Chapter 3. The nano‐fibrous sheets comprise nano‐fibers within similar range to collagen fibers, which is the main component of the ECM. The suitability of these nano‐fibrous PLLA scaffold sheets is evaluated regarding cell attachment and morphology.
Chapter 5 deals with the development of micropatterned porous scaffold sheets based on flexible poly(tri‐methylene carbonate) (PTMC). Poly(ethylene oxide) PEO addition to the casting solution plays an important role in the phase separation process and the obtained porous morphology, and this role is discussed in detail. C2C12 attachment, proliferation, differentiation and organization on the PTMC porous sheets are systematically studied.
Ch ap te r 1
Chapter 6 and Chapter 7 focus on scaffold‐cell interaction and up‐scaling. Chapter 6 presents a detailed study of the role of material properties and surface topography on cell behavior. Protein adsorption to scaffold surfaces determines cell behavior on these surfaces, and it is thought that for instance wettability of a surface plays an important role in this. Applying a micropillar array to a selection of three polymers enables altering the surface wettability of these polymers without variation in the surface chemistry. Evaluation of protein adsorption to these surfaces and correlation to cell attachment, proliferation and cell morphology is described.
Chapter 7 deals with complexity involved in tissue engineering and more specific, scaffold design. Proper functioning of the human body requires a full set of highly complex functions from single cell to complete tissue levels. Translating this complexity into the design of in vitro tissue engineering brings about a tremendous number of variations, and grows to virtually innumerable considered that optimal design is fully subject to the specific application. High‐throughput screening of a library comprising a multitude of surface topographical variations, presented in this chapter, may give a first indication of the most suitable surface designs for a specific TE application.
The work in Chapter 8 relates to scaffold architecture, going 3D and nutrient supply, herewith following up on the work described in Chapter 3 by studying the development of 3D multi‐layer scaffolds. This chapter comprises the evaluation of nutrient transport throughout the 3D scaffold under both static as well as dynamic conditions and the resulting effect on cell behavior. Besides, a theoretical model is designed and validated to predict optimal use of the multi‐layer scaffold by introduction of nutrient perfusion throughout the scaffold.
Chapter 9 reflects back on the results of this thesis and presents an outlook on a number of topics to be explored in the future to improve and optimize the 3D multi‐layer scaffold as proposed in this thesis.
References
1. Langer R, Vacanti JP. Tissue Engineering. Science 1993;260:920‐926.
2. Vacanti CA. History of Tissue Engineering and A Glimpse Into Its Future. Tissue Engineering 2006;12(5):1137‐1142.
3. Fuchs JR, Nasseri BA, Vacanti JP. Tissue engineering: A 21st century solution to surgical reconstruction. Annual Thorac Surgery 2001;72:577‐591.
4. Lanza RP, Langer RS, Vacanti J. Principles of tissue engineering. 2nd ed. San Diego, CA :: Academic Press, 2000.
5. Saltzman WM. Tissue Engineering: principles for the design of replacement organs and tissues. 1st ed. Oxford: Oxford University Press, 2004.
6. Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials 2007;28(25):3587‐3593.
7. Blitterswijk CAv, Thomsen P. Tissue engineering. 1st ed. Amsterdam; Boston: Elsevier/Academic Press, 2008.
8. Baud V, Jacque E. The alternative NF‐kappa B activation pathway and cancer : friend or foe? M S‐Med Sci 2008 Dec;24(12):1083‐1088.
9. Chen Y, Alman BA. Wnt Pathway, an Essential Role in Bone Regeneration. Journal of Cellular Biochemistry 2009 Feb 15;106(3):353‐362.
10. Landmesser U, Wollert KC, Drexler H. Potential novel pharmacological therapies for myocardial remodelling. Cardiovasc Res 2009 Feb;81(3):519‐527.
11. Hollister SJ. Porous scaffold design for tissue engineering. Nature Materials 2005;4:518‐524. 12. Takezawa T. A strategy for the development of tissue engineering scaffolds that regulate cell behavior. Biomaterials 2003;24(13):2267‐2275.
13. Cao YMDPD, Vacanti JPMD, Paige KTMD, Upton JMD, Vacanti CAMD. Transplantation of Chondrocytes Utilizing a Polymer‐Cell Construct to Produce Tissue‐Engineered Cartilage in the Shape of a Human Ear. Plastic & Reconstructive Surgery 1997;100(2):297‐302.
14. Fong P, Shin'oka T, Lopez‐Soler RI, Breuer C. The use of polymer based scaffolds in tissue‐ engineered heart valves. Progress in Pediatric Cardiology 2006;21(2):193‐199.
15. Jawad H, Ali NN, Lyon AR, Chen QZ, Harding SE, Boccaccini AR. Myocardial tissue engineering: a review. Journal of Tissue Engineering and Regenerative Medicine 2007;1(5):327‐342.
16. Nesic D, Whiteside R, Brittberg M, Wendt D, Martin I, Mainil‐Varlet P. Cartilage tissue engineering for degenerative joint disease. Advanced Drug Delivery Reviews 2006;58(2):300‐322. 17. Matsumura G, Hibino N, Ikada Y, Kurosawa H, Shin'oka T. Successful application of tissue engineered vascular autografts: clinical experience. Biomaterials 2003;24(13):2303‐2308.
18. Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, et al. Clinical transplantation of a tissue‐engineered airway. The Lancet 2008;372(9655):2023‐2030.
19. Stamatialis DF, Papenburg BJ, Gironés M, Saiful S, Bettahalli SNM, Schmitmeier S, et al. Medical applications of membranes: Drug delivery, artificial organs and tissue engineering. Journal of Membrane Science 2008;308(1‐2):1‐34.
Parts of this chapter are included within a review paper: D.F. Stamatialis, Bernke J. Papenburg, M. Gironés, S. Saiful, S.N.M. Bettahalli, S. Schmitmeier, M. Wessling
2.1 Requirements scaffold
Within tissue engineering (TE), one of the major research themes is scaffold design. A scaffold is a 3‐ D construct that serves as temporary support for isolated cells to grow into new tissue before transplantation back to the host, as described in Chapter 1. The design of the scaffold determines the functionality of the construct to a high extent. Although the final requirements depend on the specific purpose of the scaffold, several general characteristics and requirements need to be considered for all designs [1‐4]. The scaffold should be/have:
- biocompatible; the scaffold should provoke an appropriate biological response in a specific application and prevent any adverse response of the surrounding tissue [5, 6]
- biodegradable; the scaffold materials should degrade in tandem with tissue regeneration and remodeling of the extracellular matrix (ECM) into smaller non‐toxic substances without interfering with the function of the surrounding tissue [7]
- promote cell attachment, spreading and proliferation; vital for the regulation of cell growth and differentiation [8]
- suitable mechanical strength; its strength should be comparable to in vivo tissue at the site of implantation as evidently, a scaffold requires more flexibility or rigidity depending on the application in e.g. cardiovascular versus bone prostheses [9]
- good transport properties; to ensure sufficient nutrient transport towards the cells and removal of waste products the scaffold should be highly porous with good pore connectivity, however, it should maintain sufficient mechanical strength implying optimization of porosity [1, 10‐12] - easy to connect to the vascularization system of the host; to ensure good nutrient supply
throughout the scaffold post‐implantation, the scaffold should be connected to the natural nutrient supplying system [1, 10, 13]
- suitable surface characteristics; apart from optimal physiochemical properties, research suggests that the introduction of e.g. surface topography into the scaffold improves tissue organization leading to increased tissue function [14‐17]
2.2 Materials
2.2.1 Common biomaterials
Due to the variation in mechanical properties required in ‘soft’ versus ‘hard’ TE applications, the constructs for these two sub‐categories generally use different classes of biomaterials. For soft TE applications, e.g. skeletal muscle or cardiovascular substitutes, generally a wide variety of polymers are applied. On the other hand, hard tissue replacements, e.g. bone substitutes, are generally based on more rigid polymers, ceramics and metals. Frequently used biomaterials originate from a wide range of natural as well as synthetic sources. It is beyond the scope of this chapter to discuss all sources in detail. This section only describes a selection of widely used materials and focuses more on the materials used in this thesis. For in‐depth information on other frequently used materials for scaffold fabrication, excellent reviews are available [10, 11, 18‐20]. Table 1 lists polymers extensively applied in scaffold fabrication for ‘soft’ TE applications. Apart from single polymers, scaffolds are also commonly fabricated from co‐polymers of two or more polymers (not listed) to improve the overall characteristics; co‐polymers generally have an average of the mechanical properties of the incorporated single polymers. Table 1 Materials frequently applied in soft TE applications. Origin Polymer (family) Collagen component of the extra cellular matrix ‐ ECM Fibrin Gelatin Poly(hydroxybutyrate) Natural Polysaccharides most common are hyaluronic acid, chitosan, starch and alginates Poly(esters) most common are poly (α‐hydroxy acids): poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) Poly(ε‐caprolactones) Poly(propylene fumarates) Poly(anhydrides) Synthetic Poly(orthoesters) Scaffold fabrication for hard TE applications employs a wider variety of classes of materials; including polymers, ceramics, composites and metals. Table 2 presents materials extensively used in hard TE, besides the polymers already listed in Table 1. Often, polymers alone might not have sufficient mechanical strength, which can be improved by adding reinforcements resulting in composites. Herewith, combining two or more classes of materials improves the mechanical properties, similar to the principle behind co‐polymer.
Ch ap te r 2 Table 2 Materials frequently applied in hard TE applications. Class of material Type Hydroxyapatite most common since it is the inorganic component of natural bone Tricalcium phosphate Crystalline ceramics Calcium metaphosphate Silica Amorphous glasses Bio‐glass Hydroxyapatite / poly(ε‐caprolactone), chitosan, and/or collagen Titanium/calcium phosphate, polyvinyl alcohol, and/or boron Composites Poly(lactic acid)/ tricalcium phosphate, silica, and/or ceramic Stainless steel Titanium Metals Alumina 2.2.2 Poly(lactic acid)
In this thesis, we primarily use one of the most common and well‐known biomaterials: poly(lactic acid) (PLA). PLA belongs to the polyester family, as is the case for the vast majority of biodegradable polymers. PLA exists in different isomeric forms, namely semi‐crystalline D(‐) (PDLA), semi‐crystalline L(+) (PLLA) and amorphous racemic D,L (PDLLA) [21, 22]. Figure 1 Molecular structure of PLA.
PLA degrades by bulk hydrolysis and leads to the production of lactic acid. In case of PLLA, degradation results in L(+) lactic acid, a substance that exists in the human body under natural circumstances as well, therefore PLLA is generally preferred over PDLA [21]. The body transports the produced L(+) lactic acid to the liver, converts it into pyruvic acid and upon entering the tricarboxylic acid cycle, secreting it as water and carbon dioxide [10].
Despite the FDA‐approval of PLLA and the large number of clinical applications, a number of literature studies report inflammatory responses [23, 24]. During degradation, the produced lactic acid can lower the pH in the environment adjacent to the polymer. This local acidity can aversively affect cellular function [25] and induce inflammatory response [26]. Additionally, highly crystalline parts might stay behind which can cause an inflammatory response of the surrounding tissue. However, it was also noted that in case of relatively small material volume, no adverse biological
responses occur. In addition, other literature reports that PLA does not leave significant amounts of accumulating degradation products behind in the body.
The degradation of PLLA in vitro occurs in the order of years, whereas in vivo degradation takes approximately 8‐10 months; degradation of PDLLA is in the order of months [1, 27]. The degradation rate of PLA scaffolds highly depends on amongst others molecular weight and polydispersity of the polymer, process parameters and scaffold design [1].
PLLA exhibit superior mechanical strength compared to PDLLA due to its semi‐crystalline nature (10‐ 40 % crystallinity) and higher Tg of around 65 °C versus around 54 °C for PDLLA [28]. Therefore,
mostly PLLA is selected over PDLLA as scaffold material, as is also the case for the vast majority of the work presented in this thesis.
2.2.3 Poly(ε‐caprolactone)
Another polymer selected of the polyester family is poly(ε‐caprolactone (PCL), a semi‐crystalline rubbery polymer with a very low Tg of around ‐60 °C [21]. Generally PCL degrades by bulk hydrolysis
like PLA, although also enzymatic degradation can occur under certain conditions. Degradation is significantly slower compared to PLA due to limited fluid inflow as result of the close packed macromolecules; in vivo degradation time extents to over 2 years [1, 29]. Therewith, PCL is mainly suitable for long‐term implants.
2.2.4 Poly(tri‐methylene carbonate)
Poly(tri‐methylene carbonate) (PTMC) is another rubbery material with high elasticity which can be attractive in certain soft TE applications. Amorphous PTMC exhibits a Tg of around ‐15 °C. High
molecular weight PTMC yields relatively good mechanical properties [30]. Figure 2 Molecular structure of PTMC. PTMC hardly degrades in aqueous solutions, whereas it degrades in the order of weeks via enzymatic degradation in vivo. Degradation of PTMC does not lead to local decrease of pH in the surrounding tissue of the scaffold, as in the case of e.g. PLA [31, 32]. Chapter 5 is dedicated to scaffold design based on high molecular weight PTMC.
Ch ap te r 2 2.2.5 Poly(ethylene oxide)/poly(butylene terephtalate)
This copolymer consists of hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(butylene terephtalate) (PBT) segments. Variation in the composition of the PEOT/PBT copolymers allows tailoring of the mechanical, biological and physicochemical properties of the material [33]. Herewith allows PEOT/PBT application in a range of TE constructs, of both soft as well as hard TE origin [34]. PEOT/PBT is well studied as bone filler material, due to the bone‐bonding character of the copolymer (especially with a high PEO content) [35‐37]. Degradation occurs upon hydrolysis and oxidation and is in the order of months, depending on its composition [33, 35, 36]. 2.2.6 Poly(dimethyl siloxane) Poly(dimethyl siloxane) (PDMS) is extensively used in microfluidics and “lab on a chip” applications as it is easy processable, cheap and transparent offering the opportunity of easy imaging. In the past 10–15 years, there has been an increased interest in the use of microfluidics in TE. The lab on a chip approach allows scientists to control the accuracy of tests, perform high throughput screening of biomaterials regarding cell response or biological reactions in general [38‐42]. As these fields more and more expand to biomedical applications, often PDMS is selected within specific studies related to these disciplines [43]. Beneficial is the high gas permeability of PDMS which can be exploited for O2 supply and CO2 removal during cell culture. However, thin PDMS sheets have relatively poor
mechanical strength and often needs to be coated with e.g. fibronectin to allow good cell attachment.
2.3 Fabrication methods
A great variety of well‐known fabrication techniques are used in scaffold design for TE applications. This section briefly describes frequently applied techniques, with in the end special attention to polymer casting and phase separation as these are the main fabrication methods used in this thesis.
2.3.1 Emulsion freeze‐drying
In emulsion freeze‐drying, homogenization of a polymer–solvent system and water leads to formation of an emulsion [44, 45]. An emulsion exists of two phases, a continuous phase and a dispersed phase within; here, the continuous phase consists of the polymer‐rich phase, whereas water is the dispersed phase. The emulsion is cooled down quickly to freeze the solvent and water, resulting in solidification of the polymer directly from the liquid state and the creation of a porous polymer structure. Subsequently, the frozen solvent and water are removed by freeze‐drying. Emulsion freeze‐drying is attractive for creation of relatively thick scaffolds with large pores. Additionally, incorporation of proteins is enabled during the fabrication of the scaffold. The obtained morphology is mainly non‐percolated (solid‐wall like pores), which is the major drawback of freeze‐ drying as this often limits cell in‐growth and nutrient transport through the scaffold.
2.3.2 Foaming
In general, foaming uses a soluble inert gas, e.g. CO2 or N2, in the supercritical region as blowing
agent to create porosity in polymers via pressure quenching [46‐48]. Variation of the process conditions enables tuning of the scaffold properties [49]. Instead of using a single polymer, this method is also applicable for composites of polymer and (bio)ceramic to employ in hard TE constructs [50]. Beneficial is the lack of solvent, eliminating the risk of remaining residues, and the low processing temperatures preventing degradation of the polymer during processing. The scaffolds often have a closed surface (skin) and mainly non‐percolated pores which can be a serious drawback of the method as these characteristics limit nutrient transport through the scaffold. Nonetheless, it is possible to obtain open porous morphologies in particular cases [51‐53]; however, the pore size is often too small for TE applications. Through additional post‐processing steps, interconnected pores can be introduced by, for example, plasma treatment or pulsed ultrasound to break the walls of the non‐percolated pores [48].
Ch ap te r 2 2.3.3 Particle leaching
Particle (or particulate, salt, porogen) leaching combines with various different techniques such as solvent casting [54, 55], compression‐molding [56] or foaming [46, 50]. Particle leaching incorporates particles, e.g. salt, sugar or specifically prepared spheres, dissolved in a polymer sample and subsequently washed out after processing the polymer sample into the final form creating (additional) porosity in the scaffold. The biggest advantage of particle leaching is the creation of scaffolds with big pores, well‐controlled high interconnected porosity and pore morphology. However, the method is not applicable for all materials such as soluble protein scaffolds and additionally, it may be a time‐consuming post‐processing method with the risks of remaining residues after processing.
2.3.4 Electrospinning
Electrospinning (ESP) is based upon charging of a polymer solution and subsequent ejection through a capillary tip or needle [57, 58]. The jet coming from the needle draws towards a collector due to an electric field ranging from 10 to 30 kV. Evaporation of the solvent from the jet after leaving the needle results in fiber deposition on the collector. To obtain continuous fibers, the method requires using solutions containing relatively high polymer concentrations of usually around 10‐15 wt%. Rotating the collector creates a non‐woven mesh with a preferential orientation of the fiber. The diameter of the fibers is within the range of nanometers to microns. Varying the process parameters, e.g. strength of the electric field, distance between needle–collector, polymer concentration, allows tuning of the fiber diameter [36, 59]. Figure 3 presents an example of an electrospun scaffold from PEOT/PBT (reprinted from [36]) with permission from Elsevier). A major advantage of electrospinning is the high flexibility and fiber resolution of the obtained scaffold. Additionally, alignment of the electrospun fibers is enabled to induce cell and tissue alignment [60]. A drawback of electrospinning is the risk of breaking fibers during fabrication, which might lead to inferior quality of the scaffold.
Figure 3 Typical SEM image of a PEOT/PBT 300/55/45 electrospun scaffold (printed from [36] with permission from Elsevier). 2.3.5 Sintering Sintering refers heat‐treatment of a powder to make the particles adhere to each other. Application of scaffolds fabricated by sintering is mainly in hard TE constructs. Traditionally, sintering uses ceramic powders; however, this method is also applicable for other materials such as metals, glasses and certain polymers as well as composites. In the latter, the heat‐treatment pyrolizes the polymer and the ceramic particles adhere taking over the porous design of the polymer sheet [20, 61]. The possibility of creating controlled and graded porosity is the main advantage of sintering. Detrimental is the possible risk of low interconnectivity of the pores and the brittleness of the fabricated scaffold in case of using certain materials. 2.3.6 Polymer casting and phase separation
Several fabrication methods based on polymer casting, with or without subsequent phase separation, are frequently applied to produce TE scaffolds. Methods often used for phase separation are e.g. liquid induced phase separation (LIPS, immersion precipitation) [62‐64] and thermally induced phase separation (TIPS) [65‐68]. Without phase separation, polymer solidification is generally achieved by solvent evaporation [69‐72]. These methods allow processing of pure polymers as well as composites of polymer–(bio)ceramic for application in hard TE [73]. In this thesis, generally polymer casting is performed on a micropatterned mold. In this case, due to the solidification of the polymer on the mold, the inverse micropattern is imprinted in the polymer sheet. When combined with LIPS, this technique is called phase separation micro‐molding (PSµM) [17, 74‐ 76]. The advantage of PSµM is the combination of micropatterning with porosity both in one fabrication step. Variation of the mold design enables variation in the obtained micropattern
Ch ap te r 2 whereas tuning of the process parameters allows tailoring of the sheet porosity. Figure 4 presents an example of a PLLA sheet fabricated by PSµM. Chapter 3 describes extensively the application of PSµM for scaffold fabrication.
The advantage of polymer casting is the possibility to create a wide range of porosities, pore sizes and morphologies. The major drawback of these techniques, however, is the use of organic solvents, which may leave residues after processing and therefore possibly harm the cells. Therefore, effectively washing the scaffolds prior to their contact with cells is essential. Figure 4 Typical SEM cross‐section image of a PLLA sheet prepared by PSµM. The sheet is prepared of a 5 wt% PLLA‐dioxane solution using isopropanol at 4 °C as the non‐solvent. The sheet was prepared on a mold featuring 30 µm wide continuous channels. The bar in the image represents 10 µm.
2.4 Scaffold design
The design of a scaffold ultimately determines the functionality of the grown tissue. Scaffold design comprehends the material and method used, and additionally the appearance of the construct, i.e. shape, size and surface topography. Applying surface topography in the form of a micropattern can control the behavior of attached cells; tailoring the architectural design of the micropattern can tune the impact on tissue organization [77, 78]. Mimicking the in vivo micro‐architecture around cells can improve the functionality of the growing tissue [79]. Figure 5 shows an example of cultured C2C12 mouse pre‐myoblast cells on a micropatterned PLLA sheet as presented in Figure 4. The arrow indicates the channel direction; clearly the cells align well within the micropatterned channels.
Figure 5 Confocal fluorescence microscopy image of 4 day C2C12 cell cultures on porous PLLA sheets featuring 30 µm wide channels (cell density 25 000 cells/cm2). Magnification 63x, cytoskeleton labeled with Bodipy phallacidin (green) and nucleus labeled with Hoechst (blue). The direction of the channel is indicated by the arrow.
Certain fabrication methods allow fixation of the final scaffold shape and size during processing. Others methods need post‐processing steps to obtain the scaffold structure. One way is a multi‐layer based design that obtains final 3D shape and size via lamination of stacked 2D layers [80, 81]. First 2D sheets are fabricated using one of the methods described previously. Subsequently, these sheets are stacked together and laminated using heat or chemical adhesion products, e.g. solvent of the material (see Chapter 3, [17]). Benefit of a multi‐layer approach is that it allows the introduction of surface topography throughout the 3D‐scaffold.
Ch ap te r 2
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